Phytotoxicity and Identification of Active Compounds from Elaeocarpus floribundus Blume Plant for Controlling Weeds

Phytotoxic compounds isolated and identified from different plants have the ability to use as plant-based herbicides. Phytotoxic chemicals may be essential to weed management and environmental protection in order to reduce the indiscriminate use of synthetic pesticides. It has been reported that Elaeocarpus floribundus plant possesses phytotoxic compounds. The leaf extracts of this species demonstrated significant growth inhibition against the tested plants (dicot plant lettuce and plant monocot timothy) and inhibition was dose- and species-dependent pattern. Two phytotoxic compounds were separated using different purifications methods and identified as compounds 1 and 2. All phytotoxic compounds displayed potent growth limitation against the tested species (cress). The compound concentrations needed for the inhibition of 50% growth (IC50 value) of tested species ranged from 1.06 to 8.53 µM (micromolar). Findings of this research suggest that these compounds might be responsible for the phytotoxicity of Elaeocarpus floribundus plant. The results of this study may be helpful for the development of natural herbicide to control weeds.


Introduction
In the past few years, with increasing of the globalization and international trade, dissemination of the invasive weeds has increased, posing a severe menace to the agricultural output and environmental ecosystems [1].Changes in the climate are partially responsible for the spread of exotic plant species over the world and the breakdown of traditional community and social structures [2].Te IPBES (International Plan for Biodiversity and Ecosystem Services) 2020 report stated that invasive weeds are considered a signifcant factor in the degradation of biodiversity and the cause of serious health problems for humans and animals [3,4].Te species composition and variety of diferent species are altered by spreading of various weeds.Diferent weeds also reduce the productivity and efectiveness of the invaded areas by displacing native species quickly [5][6][7].Lower crop net output worldwide may be caused by a number of issues, including low agricultural seed quality, the application of traditional and incorrect sowing techniques, inappropriate planting times with inadequate doses, insufcient input use, inadequate irrigation, and inefective or nonefective weed control methods.However, most farmers ignore weed infestations that can directly impede crop development and signifcantly reduce grain and straw yields [8].Naturally occurring competition between weeds and crops for space, moisture, nutrients, and light results in a considerable decrease in agricultural yield [9,10].Weed plants might also serve as a host to numerous insects and pathogens that harm diferent crop species.A study found that weed infestation caused a 13.2% yearly loss in crop yield globally, which is equal to thousand million people's annual provisions supply [11].Some characteristics associated with weeds include long-lasting seeds, a rapid ability to germinate, a quick growth habit, the ability to survive in unfavorable environments, and the lack of a requirement for specifc environmental conditions for emergence.Tese characteristics help the weed make a harmful obstacle for crop production.Hence, weed management is essential or performs a pivotal role for crop protection and food supply.
Tere are some traditional weed management strategies such as manual, mechanical, chemical, and biological [12][13][14].Manual weed control methods are the best way to manage weeds in a crop feld, but the cost and manpower requirements are very high to make it practical for widespread crop cultivation.Chemical control of weed is a very popular and common weed control strategy, mainly depending on the application of synthetic herbicides.Te indiscriminate use of synthetic herbicides or chemicals has led to several issues, such as environmental pollution, residues of synthetic herbicides, and weed resistance.In afuent nations, agriculture primarily relies on synthetic herbicides to manage weeds.However, since the 1980s, herbicides without a novel MOA have been launched (mechanism of action) [15][16][17].Te intensive use of synthetic herbicides without novel mechanisms of action has led to the evolution of resistance in 513 biotypes of 267 weed species against 21 diferent herbicides worldwide, across several cropping systems [18].Te USA (United States of America) has the majority of herbicide resistance cases, with over 160 plant species afected.Heavy herbicide use also has several additional negative efects, including increased chemical prices, the risk of groundwater contamination from runof and leaching, and issues with irrigation water recycling [19].Concerns among the general public about herbicides' efects on the environment and public health are also growing.
Te necessity to create an eco-friendly, long-lasting tool or instrument for weed control has arisen due to the development of synthetic herbicide-resistant weeds, stemming from the absence of new synthetic herbicides with novel modes of action and increased awareness among people about the efects of herbicides [20].Biological weed control is one of the important methods to control weed through the application of allelopathic compounds (allelopathy) which are isolated from natural sources mostly from diferent plants [21].Biological weeding strategy on the basis of allelopathy is considered one of the best weed control methods [22].Te term "allelopathy" refers to a well-known biological method involving the secretion of allelochemicals (secondary metabolites) by plants or the decomposition of plant products discharged into the soil and environment [23,24].Diferent allelochemicals such as phenolic substances, fatty acids, terpenes, sterols, alkaloids, essential oils, and saponins are necessary for a plant's interaction with its environment but are not essential for the development and growth of the plant [25].In the context of a natural ecosystem or environment, allelopathy happens when allelochemicals generated by one plant are dispersed onto nearby plants in similar population.Te allelochemicals impact their development, germination, survival, and reproduction.Several allelopathic compounds that have been identifed from diferent plants have been investigated in relation to their potential use in weed management.
Most of them already proved their harmful activities against weed species by suppressing germination, shoot, and root growth and also hampering water, nutrients uptake, and photosynthesis [26][27][28].Natural substances with allelopathic properties often have a short half-life, so there is no need to worry about residues in the soil.Various studies claim that the natural substances or allelochemicals of particular plant species could play a vital role in weed management tactics due to this beneft as well as their efectiveness [29,30].Te application of extracts from various plant species, rich in natural compounds that have phytotoxic efects against weeds, might reduce the use of synthetic herbicides.Additionally, it could protect humans from health hazards and save the environment from pollution [31].Tus, the use of allelochemicals for controlling weeds may be of great importance in reducing the indiscriminate application of synthetic herbicides.
Te tree Elaeocarpus foribundus Blume, locally known as jalpai in Bangladesh, is one of the most common medicinal species in Bangladesh under the Elaeocarpaceae family.Tis species is a nondeciduous tree, generally cultivated in the Asian regions of East and South Asia, such as India and Bangladesh.Te plant species are grown in different areas of the world such as Australia, China, Fiji, Madagascar, Hawaii, Japan, Mauritius, Tailand, and Malaysia [32,33].Tis tree is found in the eastern Himalayan region at heights of over 900 meters, in the northern region of Kanara's nondeciduous forest, and in the western part of coastal areas.In India, Elaeocarpus foribundus is found in diferent regions such as Jalpaiguri, Alipurduar, the northern parts, and Bihar.It is believed that Jalpaiguri is a district of India originated from the words "jalpai," which means "the plant of jalpai," and "guri," which means "plant trunk" [34].Tis tree is an underutilized and unexploited fruit plant that grows in homestead areas with little to no maintenance.Te size and shape of the fruit are similar to olive fruit; hence, it is named Indian olive or jalpai in Bangla.Elaeocarpus foribundus is a medium-to large-sized tree that bears simple, alternate leaves.Sometimes, some of the leaves turn red or orange, which is a very important feature of this tree.Te fowers of this tree start to appear in the months of April to May, and the fruits mature from August to October.Te fruit is a drupe, greenish in color, with the edible portion, the mesocarp, found around the seed.Both immature and mature fruits have a sourish favor and are commonly used to prepare pickles and chutney.Te bark and leaves are typically used for mouthwash, and the fruits also possess antiseptic properties.Te fruits have the ability to decrease and fx the activities of Ag (silver) biosynthesis, displaying a growth inhibition efect against both Grampositive and Gram-negative bacteria [35].Tis tree wood has diferent usages including plywood, fberboard, constructions, and furniture.Many parts of this plant, including the roots, barks, leaves, and fruits, are traditionally used to treat a variety of diseases.
Te plant has several pharmaceutical properties those are used for the treatment of various disorders like arthritis, high pressure, diabetes, and dysentery [36].Besides, this plant is also very famous for possessing diferent biological efects including antiaging, anticancer, antitumor, antiseptic, antioxidant, and antibacterial [37,38].Tere have been many studies on the plant's various therapeutic applications and other uses, and we expect that it may contain allelopathic properties as well as allelopathic compounds.Terefore, current research was carried out to isolate and characterize phytotoxic compounds by using several isolation steps from Elaeocarpus foribundus for controlling weeds.

Research Plant Species. Te Elaeocarpus foribundus
species leaves were assembled from several regions of NSTU (Noakhali Science and Technology University), Bangladesh (22 °47′ 31″N and 91 °06′ 07″E) in the months of April and May, 2019.Te collected leaf samples were cleaned under running water for removing debris, dirt, and diferent contaminants.Te cleaned leaves were placed in a shady location for drying.After desiccation, these leaves were powdered using a blender.Te leaf powder was then stored in a polybag and placed in the refrigerator before use.Two plant species, lettuce (Lactuca sativa L.) (a monocot species) and timothy (Phleum pratense L.) (a dicot species), were selected for determining phytotoxic activity through a bioassay experiment.

Extraction Procedure and Growth
Assay.Te extraction bioassay study was carried out to determine phytotoxic activity of Elaeocarpus foribundus and developed an exact isolation technique: 60 g leaves were extracted using 380 mL methanol (70% aqueous MeOH) for the time of 48 h and fltered by a flter paper (No. 2, 125 mm; Toyo Ltd., Tokyo, Japan).Te remaining residues were once again submerged in an equal amount of methanol for 24 hours and then refned.Te extracted residues were combined and dried using a rotavapor at 40 °C.Te dried leaf residues were dissolved in 140 mL of methanol to prepare diferent assay concentrations, such as 0.001, 0.003, 0.01, 0.03, 0.1, and 0.3 g dry weight equivalent extract per milliliter.Tese concentrations (6) were then applied onto a flter paper (No. 2, 28 mm; Toyo Roshi Ltd., Tokyo, Japan) in 28 mm Petri dishes.After the extracted residues were dried, the Petri dishes were wetted using 0.6 mL of a 0.05% aqueous solution of polyoxyethylene sorbitan monolaurate (Tween 20, Nacalai Tesque, Inc., Kyoto, Japan).Ten lettuce seeds (of uniform size) and ten emerged timothy seeds were placed on each Petri dish.Te control treatment consisted only of Tween 20 without Elaeocarpus foribundus.Finally, the Petri dishes were placed in a germinator for 48 hours at a temperature of 25 °C in dark conditions, and the growth of the seedlings was assessed.

Isolation of the Active Compounds.
Isolation and purifcation of active phytotoxic compounds are briefy narrated in our preceding research [39].Te compounds 1 and 2 were separated from the silica gel column using a solvent mixture of 70% ethyl acetate (EtOAc) in hexane (n-hexane), from the Sephadex LH-20 column using a 40% aqueous methanol solution, and from the C18 cartridge (reverse-phase) using a 40% aqueous methanol solution.Subsequently, they were purifed using reverse-phase high-performance liquid chromatography (HPLC) on a 4.6 × 250 mm Inertsil ® ODS- 3 column with a particle size of 5 µm, provided by GL Science Inc., Tokyo, Japan.Te HPLC was conducted at a fow rate of 0.8 mL/min with a 25% aqueous methanol solution.Te chromatograms were detected at a wavelength of 220 nm, with an oven temperature of 40 °C and retention times of 29-34 and 47-63 minutes, respectively.Lastly, these two active substances (Compound 1 and Compound 2) (Figure 1) were identifed using high-resolution electrospray ionization mass spectrometry (HRESIMS) (performed on a Termo Scientifc Orbitrap Exploris 240 Mass Spectrometer) and proton nuclear magnetic resonance (1H NMR) (Bruker Corporation, Billerica, MA, USA).

Bioassay of the Characterized Compounds.
Two characterized substances 1 and 2 were dissolved in 5 mL methanol to prepare the diferent bioassay concentrations: 0.3, 1, 3, 10, 30, and 100 µM.Tese six concentrations were then applied onto the flter paper (No. 2, 28 mm; Toyo Roshi Ltd., Tokyo, Japan) in Petri dishes (28 mm).After the flter paper was completely dry, it was wetted using 0.6 mL of a 0.05% aqueous solution of polyoxyethylene sorbitan monolaurate (Tween 20, Nacalai Tesque, Inc., Kyoto, Japan).Ten homogenous seeds of cress and ten pre-emergence seeds of barnyard grass were placed on each Petri dish and kept in a growth chamber under constant darkness at 25 °C temperature for 48 hours.Te shoot and root lengths of the cress and barnyard grass seedlings were recorded after 48 h of growth and compared with the control seedlings.

Statistical Analysis.
All of the bioassay studies were conducted with CRBD (completely randomized block design), repeated thrice, and total assay studies were replicated twice.Collected data from assay were represented as the mean ± standard error (SE).Analysis of variance (ANOVA) was conducted using SPSS software (SPSS Inc., Chicago, Illinois, USA), and signifcant variations within diferent treatments and control studies were determined using Tukey's HSD test at a 5% probability level.Te concentrations required for 50% growth limitation (IC 50 value) of the tested species in the assay experiments were determined using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, California, USA).

Phytotoxic Efect of Elaeocarpus foribundus Leaf Extracts.
Te leaf extracts (aqueous methanolic) of E. foribundus exhibited signifcant phytotoxicity against lettuce (Lactuca sativa) and timothy (Phleum pratense) seedling growth.Te limitation of seedling growth expanded with increasing concentrations of extracts, and the growth limitation also varied within the tested species (Figure 2).In the frst concentration (0.001 g dry weight equivalent E. foribundus Te Scientifc World Journal extract/mL), the growth restriction was not signifcant, but growth suppression was signifcant at the greater concentrations.At a concentration of 0.03 g dry weight equivalent E. foribundus extract/mL, the growth of tested species' seedlings was arrested by more than 80%, except for timothy shoot growth (67.4%),compared to the growth of control seedlings.Particularly, at a concentration of 0.1 g dry weight equivalent E. foribundus extract/mL, the growth of lettuce seedlings was completely suppressed, while the growth of timothy shoots and root was restricted by 90.3% and 96.5%, respectively, compared to the growth of control seedlings.However, at the highest concentration (0.3 g dry weight equivalent E. foribundus extract/mL), the growth of seedlings from both tested species (lettuce and timothy) was completely inhibited by E. foribundus plant extracts.Te IC 50 values (concentrations needed for 50% growth suppression) of the tested plants ranged from 0.004 to 0.017 g dry weight equivalent E. foribundus extract/mL (Table 1).Tese IC 50 values also indicated that root growth suppression was greater than shoot growth limitation by E. foribundus plant extracts.

Phytotoxic Activity of Characterized Compounds.
Two characterized compounds were tested to estimate their phytotoxic activity on the examined plant species (cress) with six concentrations (Figure 4).Te results of the bioassay studies also showed that the phytotoxic activity of the characterized compounds on the examined plant growth difered signifcantly, and phytotoxicity increased with increasing concentrations of the identifed compounds (Figure 4).Te examined plant growth difered signifcantly at concentrations of 1 µM or greater than 1 µM for both compounds.At the concentrations of 30 µM, species seedling growth was completely restricted by compound 1.Meanwhile, at the same concentration, seedling growth was limited to 69.83% and 75.69% (shoot and root, respectively) by compound 2 compared to control growth.Te IC 50 values of the tested plants ranged from 1.06 to 8.53 µM (Table 2).Table 2 indicates that compound 1 exhibits greater phytotoxic potential than compound 2. Te IC 50 values also suggest that root growth is more sensitive than shoot growth to both compounds.

Discussion
In the preceding studies, we evaluated the allelopathic activity of the Elaeocarpus foribundus leaf extracts against cress (Lepidium sativum), alfalfa (Medicago sativa), barnyard grass (Echinochloa crus-galli), and Italian ryegrass (Lolium multiforum) and found signifcant growth limitation [42,43].To support previous fndings, in the present research, we have also determined the phytotoxic potentiality of Elaeocarpus foribundus leaf extracts against a crop plant (lettuce) and a weed plant (timothy).Te aqueous methanol extract of Elaeocarpus foribundus leaves notably inhibited the growth of the examined plants, and the level of inhibition accelerated with increasing concentrations of the plant extracts.Many researchers also reported those kinds of growth inhibition using diferent plant extracts: Senna garrettiana, Hyptis suaveolens, Schumannianthus dichotomus, Garcinia xanthochymus, Anredera cordifolia, Clerodendrum indicum, Paspalum commersonii, Albizia richardiana, and Cassia  Te Scientifc World Journal alata [44][45][46][47][48]. Variations in IC 50 value of Elaeocarpus foribundus leaf extracts suggested that inhibition of growth was dependent on the examined species of plant (Table 1).In various studies, it was reported that susceptibility to plant extracts difered with variation of target species, indicating diferences in translocation process, mode of actions, and absorption mechanisms of allelopathic substances across plants [39].Growth suppression potentiality of Elaeocarpus foribundus leaf extracts indicates that the plant may possess phytotoxic compounds.Te Scientifc World Journal  6 Te Scientifc World Journal Te allelopathic activity of diferent plant species extracts on the recipient species is basically an outcome of the efects of phytotoxic substances that perform as toxins to inhibit the growth of recipient species or weeds [49].It has been reported that plant extracts or allelopathic compounds afect root growth inhibition more severely than shoot growth restriction.Te greater susceptibility of root growth to diferent plant species' extracts is due to the root being the initial organ of the plant to absorb phytotoxic compounds from plant extracts.Root tissues also exhibit more penetrability to allelopathic compounds compared to shoot tissues [50].Allelopathic efects are normally estimated by determining the shoots' and roots' growth elongation.Te results of this also demonstrated that shoots' growth was less susceptible than roots' growth to the Elaeocarpus foribundus plant extracts.In the previous experiments, seven allelopathic compounds such as (3R)-3-hydroxy-β-ionone, cis-3-hydroxy-α-ionone, loliolide, (+)-dehydrovomifoliol, (−)-dehydrololiolide, 3-hydroxy-7-megastigman-9-one, and elaeocarpunone (novel compound) were identifed from the Elaeocarpus foribundus plant extracts [42,43].In our current study, another two active allelopathic substances 1 and 2 were characterized from these plant extracts (Figure 3).
Compound 1 is a molecule of isoprenoid, obtained by synthesizing the carotenoid derivatives of IPP (isopentenyl diphosphate) using the pathway of MEP (methylerythritol phosphate) in the plastids [51].It is also familiar as a plant growth regulator and plays a vital role to decrease diferent stresses such as transplanting shock, heat, salinity, water defciency, and prompt crop plants establishment [52].It enhances various physiological processes, including the closing of stomata, modulation of the root system, organization of the soil microorganism (SMO) community, triggering transcriptional and post-transcriptional gene expression, and inducing metabolic changes [53].It has been recorded to generate reactive oxygen species (ROS) during the closing of stomata [54].It has been demonstrated that this compound readily degrades in the soil, producing dihydrophaseic and phaseic acid.Alternatively, depending on its concentrations, this compound can either limit or stimulate the growth of roots or radicals in diferent plant species.Application of the compound reduces the growth rate of coleoptiles in corn.Te use of this to nondormant seed showed to restrict their emergence or germination.Te infuence of this compound may be interlinked to its normal activity as an endogenous growth inhibitor during the formation of the seed.Te growth inhibitory potentials of the compound on protoplast growth of plant species along with its signifcances were determined, and it demonstrated as a phytochemical in sugar beet and Beech (Fagus).It also limited the growth of protoplast of Cherry (Prunus yedoensis) plant at little concentrations [55].Although this compound is a very important phytohormone, some studies showed that it has phytotoxic potential [56,57].Tis compound isolated and characterized as an allelopathic compound from several plants including Imperata cylindrica, Aglaia odorata, and Macaranga tanarius.Te phytotoxicity of the compound has been well documented, but there is no document about the isolation, identifcation, and phytotoxic potential of this compound from the plant Elaeocarpus foribundus.
Te obtained results from the present study demonstrate that compounds 1 and 2 signifcantly limited the growth of cress seedling (Figure 4).Te compounds I 50 value displayed that compound 1 showed stronger allelopathic activity against cress than compound 2 (Table 2).Te phytotoxic compounds afect the growth of plants through various reactions.Te researchers reported that phytotoxic activity is measured by molecular structures, especially the number and position of diferent active groups present in the compounds [61,62].Tus, these compounds are liable for allelopathic potential of this plant species.Terefore, phytotoxic efects of Elaeocarpus foribundus leaves may provide useful information for controlling weeds as well as help develop natural herbicides.

Conclusions
Te leaf extracts of Elaeocarpus foribundus signifcantly inhibited the growth of seedlings of the tested species (lettuce and timothy), and the level of suppression difered with the concentration of extracts and the species tested.Two phytotoxic substances were separated from Elaeocarpus foribundus leaf extracts using several purifcation methods and characterized by spectral analysis, identifed as substances 1 and 2. All the compounds signifcantly inhibited the seedlings' growth of tested species cress.Te results obtained from this research indicate that these two compounds (1 and 2) are liable for phytotoxic activity of the Elaeocarpus foribundus plant.Terefore, Elaeocarpus foribundus species might be helpful for controlling weeds in an environment friendly way.To confrm the obtained results, we will conduct this research in feld condition in the future.

Figure 1 :
Figure 1: Phytotoxic activity by the compounds 1 (a) and 2 (b) on cress seedlings growth at diferent concentrations.Signifcant diferences are denoted by diferent letters at 5% level of probability.

Figure 3 :Figure 4 :
Figure 3: Phytotoxicity of the Elaeocarpus foribundus (aqueous methanolic) leaf extracts on the growth of lettuce and timothy seedlings at diferent concentrations.Te mean ± standard error (SE) for every treatment from two experiments with three replications (seedlings (10) for every replication) for each experiment (n � 60) that are demonstrated.Asterisks indicated the variations between the treatments and the control: * p < 0.05 and * * * p < 0.001 (one-way ANOVA and LSD test by post hoc).

Table 1 :
Te IC 50 values (concentrations needed for 50% growth suppression) of lettuce and timothy seedling by aqueous methanolic extracts of Elaeocarpus foribundus plant.

Table 2 :
IC 50 value (the compound concentrations needed for the inhibition of 50% growth) of tested plant seedlings by the compounds (1 and 2) from the extracts of Elaeocarpus foribundus leaf.